Sample size for collecting seeds in germplasm conservation: the case of the Lima bean (Phaseolus lunatus L.)

 The design of optimum sampling strategies integrating criteria of efficiency relevant to multilocus models and many target populations has been investigated with respect to the number of plants and the number of seeds per plant to be sampled for a Lima bean (Phaseolus lunatus L.) gene pool. This study, using five populations and six polymorphic enzyme loci, shows that the number of plants rather than the number of seeds collected per plant primarily determines the success of seed sampling, suggesting that plant number plays an essential part in maintaining the allelic multiplicity of predominantly selfing species like Lima bean. According to the results, it appears that among Lima bean populations an efficient sampling procedure is achieved by collecting 1–4 seeds from 200 to 300 plants. These sample sizes will retain 8–10 alleles, regardless of their frequencies. When we consider polymorphism at the 5% level, it is expected that sampling 10–80 plants will collect combinations of 4–8 alleles. Based on data from genetic and demographic studies, we suggest an efficient sampling scheme for Lima bean germplasm at both population and geographical levels. Received: 10 March 1998 / Accepted: 1 April 1998     

Assessment of genetic relationships between Setaria italica and its wild relative S. viridis using AFLP markers

AFLP markers were used to assess genetic diversity and patterns of geographic variation among 39 accessions of foxtail millet (Setaria italica) and 22 accessions of green foxtail millet (S. viridis), its putative wild progenitor. A high level of polymorphism was revealed. Dendrograms based on Nei and Li distances from a neighbour joining procedure were constructed using 160 polymorphic bands. Bootstrap values revealed that no specific geographic structure can be extracted from these data. The high level of diversity among Chinese accessions was consistent with the hypothesis of a centre of domestication in China. The results also showed that accessions from Eastern Europe and Africa form two distinct clusters. The narrow genetic basis of these two gene pools may be the result of local-adaptation. Received: 1 June 1999 / Accepted: 16 September 1999     

Assessment of genetic diversity in a Morus germplasm collection using fluorescence-based AFLP markers

To meet various breeding objectives and to conserve the existing genetic resources of mulberry for future use, the present study was undertaken to investigate the amount of genetic diversity and to establish the relationships between mulberry genotypes using fluorescence-based AFLP markers. Genetic diversity was estimated in 45 mulberry accessions from different eco-geographic regions of Japan and other parts of the world. Five primer combinations amplified an average of 110 AFLP markers per primer combination, ranging in size from 35 to 500 bp. A high degree of polymorphism was revealed by these combinations that ranged from 69.7 to 82.3% across all the genotypes studied. Several rare genotype-specific bands were also identified which could be effectively utilized to distinguish different genotypes. The wide range in genetic similarity coefficients (0.58–0.99) indicated that the mulberry germplasm collection represents a genetically diverse popu-lation. The phenetic dendrogram generated by the UPGMA method grouped 45 accessions into four major clusters, which was in agreement with the results from conventional methods. Clustering of some genotypes into strictly separate groups was not readily apparent and no clear interrelationships could be depicted, in spite of their different geographic origin. In addition, AFLP analysis provided sufficient polymorphism for DNA typing and contributed additional insights into the genetic structure of the mulberry germplasm. These results will help in the formulation of appropriate strategies for conservation and variety improvement in mulberry, for which little or no knowledge of genetic diversity is currently available. Received: 30 December 1999 / Accepted: 14 March 2000     

Genetic biodiversity impacts of silvicultural practices and phenotypic selection in white spruce

Forest-management practices relying on natural and/or artificial regeneration and domestication can significantly affect genetic diversity. The aim of the present study was to determine and compare the genetic diversity of the pristine old-growth, naturally and artificially regenerated and phenotypically selected white spruce, and to determine the genetic-diversity impacts of silvicultural practices. Genetic diversity was determined and compared for 51 random amplified polymorphic DNA (RAPD) loci for the adjacent natural old-growth, naturally regenerated and planted white spruce stands at each of four sites, one oldest plantation and open-pollinated progeny of 30 phenotypic tree-improvement selections of white spruce from Saskatchewan. Each of the 420 white spruce individuals sampled was genetically unique. The old-growth stands had the highest, and the phenotypic selections the lowest, genetic diversity. The genetic diversity of the natural regeneration was comparable to that of the old-growth, whereas the genetic diversity of the plantations was comparable to that of the selections. On average, the genetic diversity of the old-growth and natural regeneration was significantly higher than that of the plantations and selections. The mean percent of loci polymorphic, the number of alleles per locus, the effective number of alleles per locus, heterozygosity, and Shannon’s index was 88.7, 83.8, 72.2 and 66.7; 1.89, 1.84, 1.72 and 1.67; 1.69, 1.62, 1.53 and 1.46; 0.381, 0.349, 0.297 and 0.259; and 0.548, 0.506, 0.431 and 0.381 for the old-growth stands; natural regeneration; plantations; and open-pollinated progeny of selections; respectively. Reduced genetic diversity in the plantations and selections suggest that their genetic base is relatively narrow, and should therefore be broadened in order to maintain genetic diversity, and sustainably manage and conserve white spruce genetic resources. Received: 12 March 1999 / Accepted: 17 March 1999     

RAPD markers on seed bulks efficiently assess the genetic diversity of a Brassica oleracea L. collection

 The concept of a core collection was elaborated to fit the necessity of optimizing the management, for both conservation and use, of genetic resources in sizeable collections. This approach requires an analysis of how the genetic variability is structured among the accessions. The large number of heterogeneous populations in our collection of Brassica oleracea makes genetic diversity studies based on plant-to-plant analysis impracticable. To overcome this limitation, the variability analysis by RAPD on seed bulks was investigated for its efficiency in assessing the structure of the genetic diversity of this collection. The optimal bulk size and the bulking or sampling variation were evaluated with bulks of different size and with replicated samples. A mixture of known genotypes was also used to characterise the band detection in bulks, and to compare the plant-to-plant and the bulk methods. Forty seeds were chosen to represent each population. In such a bulk, the detection of bands depended on the proportion of the genotype they were derived from in the mixture. Intense and frequent bands were detected in the bulk with a 15% detection limit. The observed bulking or sampling variation within populations was smaller than the variation between populations, leading to an efficient separation of populations with a clustering of all samples of the same population. The distances calculated from bulk data were highly correlated with the distances based on the plant-to-plant analysis. We demonstrated that RAPD on seed bulks can be used to describe the genetic diversity between populations. Received: 27 August 1998 / Accepted: 29 September 1998     

Assessment of genetic diversity in Azadirachta indica using AFLP markers

 Genetic diversity was estimated in 37 neem accessions from different eco-geographic regions of India and four exotic lines from Thailand using AFLP markers. Seven AFLP selective primer combinations generated a total of 422 amplification products. The average number of scorable fragments was 60 per experiment, and a high degree (69.8%) of polymorphism was obtained per assay with values ranging from 58% to 83.8%. Several rare and accession-specific bands were identified which could be effectively used to distinguish the different genotypes. Genetic relationships within the accessions were evaluated by generating a similarity matrix based on the Jaccard index. The phenetic dendrogram generated by UPGMA as well as principal correspondence analysis separated the 37 Indian genotypes from the four Thai lines. The cluster analysis indicated that neem germplasm within India constitutes a broad genetic base with the values of genetic similarity coefficient ranging from 0.74 to 0.93. Also, the Indian genotypes were more dispersed on the principal correspondence plot, indicating a wide genetic base. The four lines from Thailand, on the other hand, formed a narrow genetic base with similarity coefficients ranging from 0.88 to 0.92. The lowest genetic similarity coefficient value (0.47) was observed between an Indian and an exotic genotype. The level of genetic variation detected within the neem accessions with AFLP analysis suggests that it is an efficient marker technology for delineating genetic relationships amongst genotypes and estimating genetic diversity, thereby enabling the formulation of appropriate strategies for conservation and tree improvement programs. Received: 20 October 1998 / Accepted: 28 November 1998     

Genetic diversity of Cryptomeria japonica using co-dominant DNA markers based on sequenced-tagged sites

 We have investigated the genetic diversity of 11 natural populations of C. japonica using 13 polymorphic STS markers. The average unbiased heterozygosities (H _e ), the average number of alleles per locus (N _a ) and the proportion of polymorphic loci (Pl) were 0.281, 1.93 and 76.92%, respectively. Coefficients of linkage disequilibrium were calculated, and no significant deviation was found except in four combinations – which might have occurred by chance alone. The fixation index (F _IS ) for 3 loci showed statistically significant values at the 1% level. The genetic differentiation between populations was only 0.047, and there were no clear geographical tendencies in the allele frequencies or the heterozygosities among populations. Consequently, the results from STS-based co-dominant DNA marker analysis were very similar to those from a previous allozyme study. However, the resolution of the technique is greater than allozyme analysis because many loci with high heterozygosities can be evaluated, and it is very simple. Therefore, the STS-based marker approach is very useful and convenient for population genetics and genome mapping of C. japonica. Received: 18 July 1998 / Accepted: 13 August 1998     

Towards an integrated linkage map of common bean. 4. Development of a core linkage map and alignment of RFLP maps

 Three RFLP maps, as well as several RAPD maps have been developed in common bean (Phaseolus vulgaris L.). In order to align these maps, a core linkage map was established in the recombinant inbred population BAT93×Jalo EEP558 (BJ). This map has a total length of 1226 cM and comprises 563 markers, including some 120 RFLP and 430 RAPD markers, in addition to a few isozyme and phenotypic marker loci. Among the RFLPs mapped were markers from the University of California, Davis (established in the F₂ of the BJ cross), University of Paris-Orsay, and University of Florida maps. These shared markers allowed us to establish a correspondence between the linkage groups of these three RFLP linkage maps. In total, the general map location (i.e., the linkage group membership and approximate location within linkage groups) has been determined for some 1070 markers. Approaches to align this core map with other current or future maps are discussed. Received: 10 March 1998 / Accepted: 22 April 1998     

Genetic relationships among subspecies of Vigna unguiculata (L.) Walp. based on allozyme variation

 The cowpea [Vigna unguiculata (L.) Walp.] is a morphologically and genetically variable species composed of wild perennial, wild annual, and cultivated forms that are mainly used for edible seeds and pods. In this study, genetic variation in 199 germplasm accessions of wild and cultivated cowpea was evaluated using an allozyme analysis. The results from this survey showed that wild cowpea exhibits genetic variation perfectly fitted with the existing morphological classification. The cowpea gene-pool is characterized by its unusually large size. It encompasses taxa (ranked as subspecies) that could be considered as different species considering the high genetic distances observed between accessions belonging to different taxa. These subspecies can be classified into three groups characterized by their breeding systems: perennial outcrossers, perennial out-inbreds, and inbred annuals. Allozyme data confirm this grouping. Perennial outcrossers look primitive and are more remote from each other and from perennial out-inbreds. Within this large gene-pool, mainly made of perennial taxa, cultivated cowpeas (ssp. unguiculata var. unguiculata) form a genetically coherent group and are closely related to annual cowpeas (ssp. unguiculata var. spontanea) which may include the most likely progenitor of cultivated cowpeas. Received: 15 June 1998 / Accepted: 29 September 1998     

Extending a RFLP-based genetic map of rye using random amplified polymorphic DNA (RAPD) and isozyme markers

RFLP-based genetic map of rye, developed previously using a cross of lines DS2×RXL10 (F₂ generation), was extended with 69 RAPD and 12 isozyme markers. The actual map contains 282 markers dispersed on all seven chromosomes and spans a distance of 1,140 cM. The efficiency of mapping RAPD markers was close to ten loci per 100-screened arbitrary primers. A strong selection of polymorphic, intensive and reproducible fragments was necessary to reveal individual marker loci that could be assigned to rye chromosomes. Newly mapped markers cover a substantial part of the rye genome and constitute a valuable tool suitable for map saturation, marker-aided selection and phenetic studies. A specific nomenclature for the RAPD loci mapped on individual rye chromosomes, which could be helpful in managing of accumulating data, is proposed. Received: 8 May 2000 / Accepted: 17 October 2000     

Genetic variability and structure of natural and domesticated populations of Caribbean pine (Pinus caribaea Morelet)

 Isozyme analysis of seed samples derived from natural and managed populations of the tropical pine Pinus caribaea vars ‘bahamensis’ and ‘caribaea’ was used to assess population genetic structure in its native range and to detect changes occurring during early domestication of the species. Baseline data from natural populations of the two varieties showed that populations sampled as seed are characterized by high gene diversity (mean H_e=0.26) and a low level of inbreeding ( mean F_is=0.15). A UPGMA tree of genetic relatedness among populations indicates that the two varieties represent distinct evolutionary units. Within each variety there is significant differentiation among populations, and this is greater for the more fragmented populations of var ‘bahamensis’ (F_st=0.08) than for var ‘caribaea’ (F_st=0.02). Seed from a seed orchard population of var ‘caribaea’ established within its natural range showed no change in genetic diversity but did show a reduced inbreeding coefficient (F_is=0.09) compared with its progenitor populations, suggesting a decrease in selfing and/or biparental inbreeding. A bulked seed sample from an exotic plantation of var ‘bahamensis’ in Australia displayed a large increase in the inbreeding coefficient (F_is=0.324) compared with that found in natural populations, possibly due to elevated self-fertilization. Finally, a bulked seed sample from an exotic plantation population of var ‘caribaea’ from China showed enhanced genetic diversity, an increase in the inbreeding coefficient and more linkage disequilibrium than its presumed progenitor populations. It was also genetically divergent from them. RFLP analysis of chloroplast DNA variation in the Chinese sample suggested that seeds of the related taxa P. elliottii and P. taeda, or seeds derived from hybridization with these taxa growing in the seed production area, had been included in the seed crop during harvesting. We conclude that monitoring of appropriate genetic markers may be an effective means of identifying potentially deleterious genetic changes occurring during forest tree domestication. Received: 10 August 1998 / Accepted: 8 September 1998     

Genetic diversity in Eastern U.S. soft winter wheat (Triticum aestivum L. em. Thell.) based on RFLPs and coefficients of parentage

 Genetic diversity in a set of 11 red and 11 white wheat lines from the Eastern U.S. soft wheat germplasm pool was measured using restriction fragment length polymorphism (RFLP) assay and coefficients of parentage (COP) analysis. On average, 78% of all bands revealed by three enzymes with 48 RFLP clones were monomorphic. Average pairwise genetic similarity (GS) was 0.97 when data from all enzymes were pooled. Probe Polymorphic Information Content (PIC) indexes ranged from 0 to 0.73 with a mean of 0.2. Fewer than 55% of the probes revealed any polymorphism. The frequency of polymorphism in the Eastern U.S. soft white winter (SWW) wheat gene pool was much lower than that observed in the Eastern U.S. soft red winter (SRW) wheat gene pool. SWW lines formed a single group on a dendrogram based on cluster analysis of RFLP-derived GS estimates, while SRW lines did not form a single group. COP values for all pairs of the Eastern U.S. soft wheat lines ranged from 0.02 to 0.9 with a mean of 0.21. SWW wheat lines traced to 53 ancestral lines and had an average COP of 0.51. The SRW wheat gene pool had more complex parentages (mean COP=0.15 and a total of 65 ancestral lines). COPs were correlated with RFLP-based GS for all line pairs (r=0.73, P<0.01). However, correlations between the two similarity measures were substantially lower when the SRW and SWW wheat gene pools were considered individually (r values of 0.23 and 0.28, respectively). The actual GS among unrelated lines in the U.S. Eastern soft wheat gene pool appears to be higher than that observed for unrelated landraces from Southwest Asia (0.96 vs. 0.905), suggesting that the ancestral landrace parents of this gene pool were themselves drawn from a base population where inbreeding, i.e., F, was greater than zero. Received:18 April 1996 / Accepted: 6 September 1996     

Genetic diversity of castor bean (Ricinus communis L.) in Northeast China revealed by ISSR markers

Inter-Simple Sequence Repeat (ISSR) markers were employed to analyze the genetic diversity of Ricinus communis L. in northeastern China plants. We selected ten primers that produced clear, reproducible and multiple bands for these experiments and 179 bands were obtained across 39 genotypes. Polymorphic band ratios ranged from 100% to a minimum of 78.9% with an average of 96.4% while band numbers were comprised between 13 (UBC823) and 23 (UBC856). The results obtained from UPGMA clustering dendrogram and PCoA lead to 39 distinct castor bean accessions belonging to four major groups. We found that all groups shared a common node with 66% similarity while Jaccard's similarity coefficient ranged from 0.58 to 0.92. Compatible inference was also observed from the high values of heterozygosity (Ht = 0.3378 ± 0.0218), Nei's genetic diversity (H = 0.1765 ± 0.2090), and Shannon's information index (I = 0.4942 ± 0.1872). In addition, our data reveal a Nei's genetic differentiation index (G_ST) of 0.3452 and estimated the gene flow (Nm) at 0.9482. These findings clearly suggest a genetic diversity in castor bean germplasms from various geographic origins and contribute to our understanding of breeding and conservation of castor beans.     

The use of microsatellite markers for detection of genetic diversity in barley populations

 A barley lambda-phage library was screened with (GA)n and (GT)n probes for developing microsatellite markers. The number of repeats ranged from 2 to 58 for GA and from 2 to 24 for GT. Fifteen selected microsatellite markers were highly polymorphic for barley. These microsatellite markers were used to estimate the genetic diversity among 163 barley genotypes chosen from the collection of the IPK Genebank, Germany. A total of 130 alleles were detected by 15 barley microsatellite markers. The number of alleles per microsatellite marker varied from 5 to 15. On average 8.6 alleles per locus were observed. Except for GMS004 all other barley microsatellite markers showed on average a high value of gene diversity ranging from 0.64 to 0.88. The mean value of gene diversity in the wild forms and landraces was 0.74, and even among the cultivars the gene diversity ranged from 0.30 to 0.86 with a mean of 0.72. No significant differences in polymorphism were detected by the GA and GT microsatellite markers. The estimated genetic distances revealed by the microsatellite markers were, on average , 0.75 for the wild forms, 0.72 for landraces and 0.70 among cultivars. The microsatellite markers were able to distinguish between different barley genotypes. The high degree of polymorphisms of microsatellite markers allows a rapid and efficient identification of barley genotypes. Received: 26 November 1997 / Accepted: 19 January 1998     

Molecular evidence for an Andean origin and a secondary gene pool for the Lima bean (Phaseolus lunatus L.) using chloroplast DNA

 Chloroplast DNA (cpDNA) diversity has been examined using PCR-RFLP and RFLP strategies for phylogenetic studies in the genus Phaseolus. Twenty-two species, including 4 of the 5 cultivated species (P. lunatus L., the Lima bean; P. vulgaris L., the common bean; P. coccineus L., the runner bean and P. polyanthus Greenman, the year-bean), represented by 86 accessions were included in the study. Six PCR primers designed from cpDNA and a total cpDNA probe were used for generating markers. Phylogenetic reconstruction using both Wagner parsimony and the neighbor-joining method was applied to the restriction fragment data obtained from each of the molecular approaches. P. vulgaris L. was shown to separate with several species of largely Mesoamerican distribution, including P. coccineus L. and P. polyanthus Greenman, whereas P. lunatus L. forms a complex with 3 Andean species (P. pachyrrhizoides Harms, P. augusti Harms and P. bolivianus Piper) co-evolving with a set of companion species with a Mesoamerican distribution. Andean forms of the Lima bean are found to be more closely related to the 3 Andean wild species than its Mesoamerican forms. An Andean origin of the Lima bean and a double derivative process during the evolution of P. lunatus are suggested. The 3 Andean species are proposed to constitute the secondary gene pool of P. lunatus, while its companion allies of Mesoamerican distribution can be considered as members of its tertiary gene pool. On the basis of these data, an overview on the evolution of the genus Phaseolus is also discussed. Received: 1 May 1998 / Accepted: 13 July 1998     

Assessing population genetic structure of sorghum landraces from North-western Morocco using allozyme and microsatellite markers

 The level of genetic diversity and the population genetic structure of sorghum landraces from North-western Morocco have been investigated based on direct field-sampling using both allozyme and microsatellite markers. As expected, microsatellite markers showed a much higher degree of polymorphism than allozymes, but relative measures of genetic structure such as Wright’s inbreeding coefficient F _IS and Nei’s coefficient of genetic differentiation G _ST were similar for the two sets of markers. Substantial inbreeding was found to occur within fields, which confirms that sorghum is predominantly selfing under cultivation. Most of the genetic diversity in Moroccan landraces occurs within fields (more than 85%), as opposed to among fields or among regions, a result which contrasts to those of studies based on accessions from germplasm collections. It is suggested that individual fields of sorghum constitute valuable units of conservation in the context of in situ conservation practices. Received: 8 December 1998 / Accepted: 28 December 1998     

Phylogenetic analysis in the genus Cicer and cultivated chickpea using RAPD and ISSR markers

Seventy five accessions belonging to 14 species of the genus Cicer were analysed with PCR-based molecular markers to determine their phylogenetic relationships. Eight of the species were annuals and included the Section Monocicer which contains cultivated chickpea (Cicer arietinum L.). The remaining six species were perennials (five from Section Polycicer and one from Section Acanthocicer). More than one accession per species was analysed in most of the wild species; within C. arietinum, 26 accessions including Kabuli and Desi types, were studied. RAPD analyses using 12 primers gave 234 polymorphic fragments. Variability within species was detected. A dendrogram based on the Jaccard similarity index showed that the distribution pattern of variability between species was related to both growth habit and geographical origin. An accession of Cicer reticulatum was closer to accessions of Cicer echinospermum than to the four remaining of C. reticulatum, suggesting the possibility of gene flow between species. Cluster analysis for cultivated chickpea differentiated Kabuli and Desi types but we did not detect a clear relationship between groups and the geographical origin of the accessions. Received: 5 April 2001 / Accepted: 13 July 2001     

Genetic structure of wild bean populations in their South-Andean centre of origin

 The genetic structure of wild common bean populations was studied in the South-Andean centre of origin of the species. Plants were collected from 21 populations in Argentina and genetic variability was assessed for molecular and resistance markers. Polymorphism was weak for phaseolin, the major seed-storage protein, and for RAPD markers, while a high level of polymorphism was observed for resistance to anthracnose, one of the most important diseases of common bean. For the three traits, within-population variability was important and represented between 43.6% and 67.5% of the total variation. Although among-population differentiation was significant for all the traits, no correlation was found between the population distances calculated from RAPDs and resistance. These results indicate that pathogen selection pressure may be an important factor influencing the distribution of variability within and among host plant populations. Received: 28 October 1997 / Accepted: 25 November 1997     

Genetic variability in wild cherry populations in France. Effects of colonizing processes

 Isoenzymes were used to evaluate gene diversity and genetic differentiation among six populations of wild cherry (Prunus avium L.) in France. We contrast the genetic characteristics of a population resulting from a recent colonization with those of a much older population of the same species. No significant genetic structure was observed among populations; in this respect wild cherry does not differ from other forest trees. No founder effects could be detected in the newly colonized population. To explain the results, we discuss classic explanations for the lack of genetic differentiation among populations, including balancing selection and neutral drift/migration. In order to account for the absence of founder effects, we propose a hypothesis based on the life cycle of forest trees, namely that the length of the juvenile phase reduces the impact of small numbers of initial founders. Received : 26 November 1996 / Accepted : 20 December 1996     

Mapping of three self-fertility mutations in rye (Secale cereale L.) using RFLP, isozyme and morphological markers

 Three mutations determining self-fertility at the S, Z and S5 self-incompatibility loci on chromosomes 1R, 2R and 5R of rye, respectively, were mapped using three different F₂ populations. There was a close linkage of one isozyme and four RFLP markers, and no recombinant plants were detected. These markers are Prx7, Xiag249 and Xpsr634 for the S locus (1R), Xbcd266 for the Z locus (2R) and Xpsr100 for the S5 locus (5R). Linkage data for markers associated to the self-fertility mutations at the S, Z and S5 loci were calculated and compared with genetic maps computed by MAPMAKER multipoint analysis. Received: 8 October 1997 / Acepted: 26 November 1997